The Tubulin-Based-Polymorphism Method Provides a Simple and Effective Alternative to the Genomic Profiling of Grape

Abstract

The TBP (Tubulin-Based-Polymorphism) method, based on a nuclear ILP (Intron-Length-Polymorphism) molecular marker, has been used for genotyping 37 accessions of the genus Vitis inclusive of different species, rootstocks, wild and cultivated subspecies. A distinct DNA barcode made up by a different number of amplicons, was attributed to each of the different accessions. TBP data were compared with those obtained, with the use of an internationally validated set of six SSR markers. Genetic relationships among the different accessions, dendrogram distributions, correlation values and polymorphic index values (PICs) were definitively comparable when not in favor of TBP. Such an experimental consistency is based upon a genomic organization of the multiple members of the β-tubulin gene family, the targets of TBP-mediated amplification, that is conserved in Vitis as in any other plant species. The TBP amplicons can actually be used as a useful source of sequence polymorphisms for generating primer pairs capable of identifying specific cultivars in a simple assay. An example for the identification of the 'Sangiovese' cv. is reported. More generally, these data are discussed in terms of the actual advantages that the introduction of the TBP method in the field of grape characterization and genotyping can provide.

Fig 1. TBP genomic profiles.

TBP amplification pattern of the 37 grapevine accessions of Table 1. Cvs are ordered according to cluster analysis of Fig 2. Blue boxes indicate few polymorphisms associated to clones of the same cv. (NBC vs NBR and MEM vs MR5). Arrows on top indicate the profile of the IRP2 cross and its parentage (PN1 x RI2); I and II indicate TBP-amplified products containing introns longer or shorter than 500 bp; Molecular sizes marker is reported on the left.

Fig 2. TBP derived cluster analysis.

Unweighted pair group method with arithmetic mean (UPGMA) tree based on Nei and Li’ genetic distance calculated on the TBP data of 37 grapevine accessions. The bar at the bottom of the figure provides a scale, from 0 to 1, of the estimated similarity. The numbers next to each node represent the bootstrap value estimated for 1000 replicates.

Fig 3. TBP versus SSRs comparative cluster analysis.

Dendrograms of eight selected cvs produced from either SSRs (A) or TBP (B) data. Grapevine cvs are those in common with the International consortium study [32]. The cv. code refers to the accession name reported in Table 1. The estimated similarity value, ranging from 0 to 1, is reported on bars at the top of each dendrogram.

Fig 4. Genomics of the Vitis vinifera β-tubulin loci with reference to the first intron sizes.

Vitis vinifera β-tubulin genes first intron size: comparison between nucleotide sequence information ('Pinot Noir' PN 40024) retrieved from V. vinifera Genome Data Base (Genoscope 12X—Assembly version 12 Feb 2010) and experimental data obtained with CE-TBP. The first column on the left reports the schematic representation of the genomic organization of the β-tubulin genes in Vitis. Introns, represented by lines, are interspersed within exons, represented by black boxes. The last columns on the right report the size of the amplified CE-TBP fragments and deduced 1st introns length, obtained from cv. PN5 ('Pinot Noir' 5V17). Chromosome number (Chr), strand, nucleotide number (left and right border) and the length in base pairs of the 1st exon and 1st intron are reported for each entry. Peak size, in base pair for each β-tubulin gene, is detecdet by the CE-TBP 1st intron method; estimated size of the 1st intron is calculated by subtracting 305 bp from the Peak size; nd not determined.

Fig 5. A doublet characterizes the ‘Sangiovese’ (SGM) cv.

On the left: CE-TBP values (peak size in base pair and peak height in Relative Florescence Unit—RFU) obtained from the amplification of the first β-tubulin intron, relative to entry 8 of the Vitis vinifera Genome Data Base (VvGDB, version Genoscope 12x). The box highlights the 670–680 bp specific doublet of the SGM cv., detected in all analysed clones. The SGM specific peak of about 670 bp is further highlighted by the arrow. On the right, the corresponding portion of the electropherogram are reported for each analysed cvs (‘Sangiovese’ (SGM); ‘Croatina’ (CRM); ‘Ancellotta’ (A1); ‘Lambrusco’ (L1, L3) and ‘Nebbiolo’ (NBC, NBR)). The star points to the specific 670 bp peak of ‘Sangiovese’ clones.

Fig 6. Molecular basis of the 'Sangiovese’ (SGM) cv. specific polymorphism.

(A) Sequence alignment of portions of the β-tubulin genes of three wine making cvs (‘Sangiovese’, ‘Croatina’, ‘Nebbiolo’), with the corresponding entry 8 of the Vitis vinifera Genome Data Base (VvGDB, version Genoscope 12x). Different molecular clones of the same cv. are indicated by different numbers. A 10 bp deletion is found in the SGM-17 clone. The arrows underscore the sequences used to design the forward and reverse primers and the star represents the fluorophore marker linked at 5’ position. (B) Portion of the electropherogram obtained by resolving, through capillary electrophoresis, the size of genomic DNA fragments amplified with the primer pair reported in (A). A specific doublet of 133–143 bp is detected only in the ‘Sangiovese’ (SGM) clones.